Preparation of the Plant Extracts
Fresh Chrysophyllum albidum seed cotyledon was collected from Ita-Alasa area, Ogbomoso North LGA, Ogbomoso, and authenticated by a taxonomist in person of Prof. Ogunkunle at the Department of Pure and Applied Biology of Ladoke Akintola University of Technology, Ogbomoso. With record ID Number LH0 867, The Chrysophyllum albidum seed cotyledons were air-dried at room temperature for four (4) weeks and pulverized before extraction. Dried powdered Chrysophyllum albidum seed cotyledons weighing 500g was successfully macerated in distiled water for 48hours. After this, the extract was filtered with clean muslin. The filtrate was concentrated in a rotary evaporator and then dried in a laboratory oven at 40ºC to obtain the powder extract.
LD 50
LD50 was determined at Department of Pharmacology of LAUTECH, Osogbo, Osun State. A total of seventy (70) Wistar rats of both sexes were used in the determination of the acute toxicity of the extract of Chrysophyllum abidum seed. The rats were randomly divided into seven groups of ten(10) each and the first (1st) group was given 10mg/Kg, the second (2nd) group 100mg/Kg, and the third(3rd) group 1000mg/Kg of the plant extract respectively via the oral route. They were observed for signs of toxicity, adverse effects or death. After 24hours, the remaining four(4) groups were given 2000 mg/Kg, 3000 mg/Kg, 4000 mg/Kg and 5000mg/Kg of the plant extract respectively and observations were noted as previously described. The LD50 of chrysophyllum albidum seed extract was found to be ≤ 5000mg/kg body weight according to Damilola., (13). The LD50 used was 750mg/kg body weight which showed relative significance in the study.
Experimental animals
Forty (40) male healthy Wistar rats weighing between 90g-150g were purchased from Laboratory Animal Department of Ladoke Akintola University of Technology and were used for this experiment. The experimental animals were acclimatized in the Anatomy Department, College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso for two weeks at room temperature with a 12hr light and dark cycle. They were fed with standard pellets gotten from the animal house and distilled water ad libitum.
Experimental Design
The animals (40 Wistar rats) were separated into four (4) groups with ten (10) research animals per group; Group 1: which serves as control group (normoglycemic), received with 2 mls/kg bw distilled water only, Group 2: which was Hyperglycemic group, received 2 mls/kg bw of distilled water only, Group 3: which was Hyperglycemic group, received 750 mg/Kg of the aqueous extract of Chrysophyllum albidum seed cotyledon. Group 4: which was normoglycemic group received 750 mg/kg of the aqueous extract of Chrysophyllum albidum seed cotyledon. Hyperglycemia was induced in overnight-fasted randomly selected rats by a single intraperitoneal administration of Streptozotocin (STZ) at 50mg/Kg body weight, which was dissolved in citrate buffer (0.1M, pH 4.5) just prior to injection (14). The experimental animals were administered 5% dextrose (Each 100 mL of 5% Dextrose Injection, USP, contains dextrose, hydrous 5 g in water for injection. The caloric value is 170 kcal/L. The osmolarity is 252 mOsmol/L (calc.), which is slightly hypotonic. The solution pH is 4.3 (3.2 to 6.5).) water after administration of STZ to stabilize them and prevent hypoglycemia (15). Hyperglycemia was allowed to develop for 72hours (16). Animals with fasting blood glucose ≥ 200mg/dl were considered hyperglycemic (17) and were included in this study.
Administration was by oral method using oral cannula. The extract was weighed using digital electronic laboratory weighing scale, while the distiled water was measured using 2ml/5ml syringes. The measured dose of the extract was dissolved in 2ml of distiled water and administered /kg bw to the animals. These animals were treated daily at around 9–10 hours for 3weeks.
Blood glucose and Body weight measurement
Fasting blood glucose (FBG) was monitored in overnight fasted experimental animals (the feed is being removed from their cage after they must have been fed in the evening), at 9–10 hour using samples collected from the tail tip, by means of a glucometer (ACCU-CHEK Active) and compatible blood glucose test strips. The Fasting blood glucose (FBG) was monitored weekly from the acclimatization period, before induction of hyperglycemia, and for the four weeks of treatment (18). The body weights of the animals were measured weekly from the acclimatization period till the end of the four weeks of treatment using a weighing scale.
Organ’s preparations
The animals were sacrificed on the 29th day of induction and administration. They were fasted overnight after the last dose of aqueous extract of Chrysophyllum albidum seed cotyledon, the fasting blood glucose was checked and the animals were sacrificed using the cervical dislocation method. The abdominal incisions were made and the pancreas and kidney specimens were harvested, and weighed across all groups. The pancreas and kidney specimens were fixed using Bouin’s fluid and 10% formolsaline respectively, and subjected into hematoxylin and eosin (H & E), Gomori and Masson trichrome stains.
Tissue Processing
The harvested pancreas and kidney specimens were fixed using Bouin’s fluid and 10% formolsaline respectively, to arrest autolysis. The tissues were removed and rinsed in tap water before being run through ascending grades of alcohol for dehydration. The tissues were taken through two (2) changes of xylene followed by tissue infiltration by a suitable histological wax. The tissues were transferred into two (2) changes of bath containing molten paraffin wax in a chamber of automated embedding machine for 1–2 hours each. The embedding was performed in special container called cassette which help to give shape to the wax containing the tissue when it is set. The tissue blocks were removed and air dried until the wax became solidified followed by sectioned with a microtome machine. The tissue samples were stained with hematoxylin and eosin (H & E) routine staining, Gomori and Masson trichome special staining to demonstrate the histoarchitecture of the tissue samples. Digital micrographs of the pancreas and kidney were obtained to show the histological and morphological changes that occurred in the treated rats as compared to the control groups. The photomicrographs were carried out with the aid of a Olympus light microscope x400, at the Department of Anatomy, Ladoke Akintola University of Technology, Ogbomoso, Oyo State.
Statistical analysis
All data were expressed as Mean ± SEM. Results were expressed as mean ± SEM and the difference between mean values was analyzed using one – way analysis of variance (ANOVA). Values of p < 0.05 were considered significant.